Gc-globulin (also designated vitamin D-binding protein) is an important plasma protein with a concentration in human plasma of approximately 300-350 mg/l (Haddad, 1995).
The daily synthesis of Gc-globulin has been determined to be 10 mg/kg bodyweight and the exchangeable pool of Gc-globulin has been estimated to be 2.9 g (80 kg bodyweight) (Kawakami et al., 1981). This gives an estimated half-life for Gc-globulin of approximately 2 days. Gc-globulin has a molecular mass of approximately 51 kDa and the complete structure of Gc-globulin has been determined (Cooke and David, 1985).
Gc-globulin is an actin-binding protein (Baelen et al., 1980), and is a part of the plasma actin-scavenging system (Lee and Galbraith, 1992). The plasma actin-scavenging system consists of gelsolin, which dissociates polymeric F-actin to monomeric G-actin subunits and Gc-globulin which sequesters G-actin and removes it from the circulation.
Actin is a cytoskeletal protein, which may be released into the circulation upon cellular damage as occurs in conditions like intoxications (e. g. hepatic), inflammations (e. g. septic shock), and physical tissue injury (e. g. traffic lesions). Infusion of actin has been demonstrated to cause the formation of thrombi in rats, an effect that could be prevented by preincubation of actin with Gc-globulin (Haddad et al., 1990). In the absence of sufficient Gc-globulin, actin-induced coagulation may lead to subsequent circulatory complications and multiple organ failure. In rats injected actin has been shown to form complexes with Gc-globulin and to be removed primarily by the liver with actin-Gc-globulin complexes being removed faster than actin itself (Harper et al., 1987, Dueland et al., 1991).
Plasma levels of Gc-globulin have been shown to be decreased in patients with liver cirrhosis (Barragry et al., 1978, Walsh and Haddad, 1982, Bouillon et al., 1984, Masuda et al., 1989), hepatic necrosis (Lee et al., 1985, Goldschmidt-Clermont et al., 1988), hepatic acetaminophen (paracetamol) intoxication (Lee et al., 1995, Schiødt et al., 1995), and septic shock (Lee et al., 1989). Decreased levels of free Gc-globulin and increased levels of Gc-globulin-actin complexes can be correlated to survival rate in patients with fulminant hepatic failure (Goldschmidt-Clermont et al., 1988, Lee et al., 1995, Schiødt et al., 1996, 1997, Wians et al., 1997), multiple trauma (Dahl et al., 1998), and septic shock (Lee et al., 1989). In a hamster model of acetaminophen-induced fulminant hepatic necrosis the decreased level of Gc-globulin and increased level of actin-Gc-globulin complexes correlated with the severity of the disease (Lee et al., 1987, Young et al., 1987). Similar observations have been made in rats with experimentally induced septic shock (Watt et al., 1989).
Gc-globulin has not previously been used in medicine, but Yamamoto (1994) has shown that a derivative, Gc-globulin treated with β-galactosidase and sialidase, generates a potent macrophage activating factor and in Yamamoto (1996) a recombinant Gc-globulin is described which is converted into this macrophage activating factor.
Purification of Gc-globulin from human plasma or serum has previously been described by Cleve et al. (1963), Heide and Haupt (1964), Roelcke and Helmbold (1967), Bouillon et al. (1976), Haddad and Walgate (1976), Svasti and Bowman (1978), Chapuis-Cellieretal. (1982), Haddad et al. (1984), Torres et al (1985), Link et al. (1986), Miribel et al. (1986), Taylor et al. (1986) and Swamy et al. (1995). These purifications have all been at an analytical level and are not suited for large-scale production. For example, Chapuis-Cellier et al. (1982) has described a purification procedure for one phenotype of Gc-globulin (Gc1-1) by pseudo-ligand affinity chromatography followed by gel filtration and ion exchange chromatography from 80 ml of human plasma, where the whole process is not suited for a large-scale production. Torres et al (1985) describes an analytical purification procedure for Gc-globulin from serum from one psoriasis patient using a two-step ion exchange displacement chromatography followed by removal of the carboxymethyldextran and further purification on a hydroxyapatite column. Roelcke and Helmbold (1967) describes an analytical purification procedure for Gc-globulin consisting of three column chromatography steps on the same matrix; hydroxyapatite.
Affinity chromatography on vitamin D (Link et al., 1986, Swamy et al., 1995) or actin columns (Haddad et al., 1984), or lengthy procedures including multiple column chromatographic steps have been used. For example, Cleve et al. (1963) used ammonium sulfate precipitation, ion exchange chromatography on TEAE cellulose in phosphate buffer at pH 7.2, preparative starch gel electrophoresis and size exclusion chromatography on Sephadex G-100 to obtain 23 mg Gc-globulin from 1 I human plasma. Heide and Haupt (1964) used multiple ammonium sulfate precipitations, starch gel electrophoresis and size exclusion chromatography to purify Gc-globulin from rivanol-precipitated human plasma fraction IV. Bouillon et al. (1976) used radiolabelled Gc-globulin with radioactive vitamin D in order to be able to follow it during purification and selection of fractions using DEAE cellulose chromatography, ammonium sulfate precipitation, hydroxyapatite chromatography, CM cellulose chromatography, DEAE Sephadex chromatography, repeated hydroxyapatite chromatography, Bio-Gel size exclusion chromatography, and repeated DEAE Sephadex chromatography to obtain 5.2 mg Gc-globulin from 400 ml human serum. Haddad and Walgate (1976) also used radiolabelling with vitamin D to follow Gc-globulin during purification from Cohn fraction IV. The procedure consisted of DEAE cellulose chromatography, size exclusion chromatography on Sephadex G-200, DEAE Sephadex chromatography and preparative polyacrylamide gel electrophoresis. Svasti and Bowman (1978) also used radiolabelling with vitamin D to select fractions for further purification and employed DEAE Sephadex chromatography at pH 8.3, DEAE cellulose chromatography at pH 8.8 and Sephadex G-100 size exclusion chromatography to obtain analytical amounts of Gc-globulin. Chapuis-Cellier et al. (1982) used chromatography on Affigel Blue, Sephadex G-100 size exclusion chromatography, and DEAE-Affigel Blue chromatography to obtain small amounts of Gc-globulin from plasma. Miribel et al. (1986) used sequential chromatography on immobilised Triazine dyes (Cibacron Blue 3-GA followed by DEAELAffigel Blue and finally Fractogel TSK-AF Green) to purify analytical amounts of Gc-globulin. Taylor et al. (1986) used ammonium sulfate precipitation, Blue Sepharose chromatography, DEAE-Sephacel HPLC followed by DEAE 5PW HPLC and finally size exclusion HPLC to purify mg amounts of Gc-globulin. In this procedure radiolabelling with vitamin D was also used to detect Gc-globulin.
In WO97/28688 purification of intracellular vitamin-D binding proteins (IDBPs; which are different from extracellular Gc-globulin) which are used for treating patients with over or under production of vitamin-D or other steroidal hormones. The IDBPs are purified using anion exchange chromatography, hydrophobic interaction chromatography and hydroxyapatite chromatography.
Thus, there is a need for an improved purification large-scale process for production of Gc-globulin and a Gc-globulin medicinal product.